The objectives of the proposed research are the development and demonstration of a new technique for protein sequencing which is both faster and more sensitive than presently available methods. The approach employs a new mass spectrometer inlet system recently developed in our laboratory as a liquid chromatograph-mass spectrometer system applicable to relatively involatile biological molecules. The significant feature of the "liuid-flow mass spectrometer" (LFMS) is that a flowing liquid stream can be totally vaporized, ionized, and mass analyzed allowing continuous, rapid analysis of a flowing liquid of changing composition. This instrument provides a new, and potentially powerful tool for continuous monitoring of fast reactions in solutions. In the proposed protein sequencing technique the enzymatic hydrolysis of terminal amino acids is carried out in a small reaction chamber terminated by a membrane which transmits the amino acids released by the reaction but which retains both the protein substrate and the enzyme. A continuous flow of buffer is pumped through the reaction chamber and into the mass spectrometer which continuously monitors the amino acid composition as a function of time. These data will be acquired and analyzed by an on-line computerized mass spectrometer data system to yield the linear sequence of amino acids in the protein. The major limitation on speed and sensitivity of the proposed technique is imposed by the variability in enzymatic rates depending on the nature of the amino acids adjacent to the bond; however, our preliminary studies indicate with modest success in optimizing the reaction kinetics, the technique should be capable of sequencing subnanomole quantities of proteins with total analysis times of less than one minute per residue.